Abstract Botulinum Neurotoxins (BoNTs) are a large family of protein toxins and are of great significance due to their extreme potency and the severity of the disease they cause in humans and animals. Botulism is a neuroparalytic disease of long duration, lasting up to several months. Without proper medical care, naturally occurring botulism is lethal in up to 50% of cases, and even with respiratory and other supportive care and antitoxin administration, up to 5 % of patients die. While naturally occurring botulism is rare, BoNTs are classified as a Tier 1 Category A Select Agent due to their threat as potential bioterrorist weapons. Amazingly, BoNTs are also widely used in medicine to treat more than 100 neuromuscular disorders and for aesthetic purposes. BoNTs are immunologically divided into 7 serotypes, which are further subdivided into subtypes. Today, 100s of BoNT variants have been identified by sequencing efforts, but only few have been investigated at the protein level. It is noteworthy that out of all the BoNT variants only two (BoNT/A1 and /B1) are currently used as therapeutics, and studies examining properties of other variants are infrequent. Our research groups have determined for the first time that subtypes within the BoNT/A serotype have distinct biologic properties, including cell entry kinetics, duration of action, cell and mouse toxicity, and immunological variations. This RO1 project proposes to determine on a molecular and structural level the mechanisms underlying these unique properties of A subtypes and the chimeric BoNT/FA. Specifically, this project will investigate the mechanisms underlying the shorter duration of action of BoNT/A3, the faster and more efficient cell entry by BoNT/A2, /A6, and /FA, the 1,000 fold lower toxicity of BoNT/A4. Our collaborative efforts are unique in this area, as we are able to combine detailed mechanistic studies on subdomains with cell-based and in vivo studies on the holotoxin level. The Barbieri lab will use structural modeling to guide mutagenesis studies on functional domains of BoNTs and investigate mechanisms of receptor binding and cell trafficking using neuronal cell models. The Johnson/Pellett laboratory will utilize these data to create targeted holotoxin constructs in their native hosts and conduct detailed investigations in various rodent and human cell models. Finally, based on the data from these studies, select holotoxin constructs will be investigated in mice to determine pathologic and pharmacologic consequences of structural alterations. Using this approach, we will investigate a large number of amino acid substitutions in functional domain studies and select specific alterations for the more effort- and cost-intensive construction of recombinant holotoxins. By utilizing several cell models before conducting in vivo studies, we are able to reduce the number of required animals and also use human specific models. Finally, the combination of in vitro, cell-based, and in vivo studies will provide novel insight into the mechanisms underlying the observed pathologic and pharmacologic properties of these toxins.